Chiron Corporation, Emeryville, Calif. identified the gene of HCV in 1988. The genome sequence of HCV including about 9,500 bases has been identified, and it has been revealed that HCV has a single strand RNA as its genome. HCV is often accompanied with mutation in its gene sequence, is classified as four to six subgroups based on gene sequences in regions accompanied with frequent mutation, and is further classified as, for example, subtypes 1a, 1b, 2a, 2b and 3a (P. Simmonds, et al., J. Gen Virol., 74, 661-668 (1993))
Only two- to three-tenths of subjects infected with HCV have subjective symptoms such as malaise, and most of them undergo chronic inflammation without subjective symptoms and may further undergo liver cirrhosis and liver cancer. It is difficult to detect HCV at initial stage of HCV infection by immunoassay using antibodies and to prevent the symptoms from becoming worse and/or the infected blood completely from contaminating into blood for transfusion.
Recent years, however, have seen significant advances as a result of intensive studies and have enabled the detection of HCV infection and genotyping of HCV even at initial stage of HCV infection by a genetic test in which a gene is amplified. The genotyping enables determination of the incidence rate of acute hepatitis, the rate of becoming chronic and the possibility of shifting from hepatitis via liver cirrhosis to liver cancer, enables the prediction of efficacies of administration of an interferon selected depending on the subtype and enables the identification of contagion sources and infection route. Thus, the detection or determination of HCV by such a genetic test becomes superior to immunoassay.
Certain methods for genotyping HCV have been reported. Okamoto et al., for example, have reported a method for genotyping HCV, including the steps of preparing seven different primers using a core region of HCV as a target, and carrying out a reverse transcription polymerase chain reaction (RT-PCR) using a reverse transcriptase in combination with a suitable set of primers in J. Gen. Virol., 73, 673-679 (1992). A certain method for genotyping HCV in its NS5-region has been reported. Japanese Patent Application Laid-Open (JP-A) No. 09-75100 and F. McOmish, et al. in Transfusion, 33, 7-13 (1993) have reported a method including the steps of cleaving an amplified product of the 5′-untranslated region of HCV with a restriction enzyme and then genotyping HCV. Other methods for genotyping have been reported, including the steps of directly sequencing the amplified product just mentioned above, and genotyping HCV based on the resulting base sequence information [Germer J. J., et al., J. Clin. MiCrobiol., 37(8), 2625-2630 (1999); Holland J., et al., Pathology, 30(2), 192-195 (1998); and Doglio A., et al., Res. Virol., 149(4), 219-227 (1998)].
All the conventional oligonucleotide primers for genotyping HCV are to be amplified by PCR, and no oligonucleotide primer for genotyping HCV by isothermal amplification has been reported. This is probably because the HCV RNA has a complicated secondary structure as a result typically of intramolecular hydrogen bonds, a region that can be amplified by isothermal amplification is restricted, and it is very difficult to design specific primers capable of carrying out genotyping in the region. A method for quantitatively determining HCV by nucleic acid sequence-based amplification (NASBA), a kind of isothermal amplification, has been reported (Guichon A., et al., J. Clin. Virol., 29, 84-91 (2004)). This technique cannot determine the genotype of HCV gene although it can amplify the HCV gene. Specifically, all the conventional HCV genotyping techniques using oligonucleotide primers require complicated temperature control in PCR and invite complicated procedures, long time and high cost for the determination and are not satisfactory. Demands have therefore been made to develop oligonucleotide primers that enable genotyping of HCV by isothermal amplification.